Thelong-termgoalofthisprojectistounderstandtheroleoftypeI,semi-invariantnaturalkillerT(NKT)cellsin generating mucosal immunity against respiratory infectious diseases. NKT cells are innate-like lymphocytes that, by recognizing microbial glycolipids or microbe-induced self lipids, assert a role in infectious diseases. NKT cells are disproportionately enriched in the lung mucosa, but the role of these T cells in respiratory infectious diseases is not fully understood. The lung mucosa is a major site for Francisella tularensis (Ft) invasionthatoft-timecausesafatalinfectiousdiseaseknownaspulmonarytularemia.Ftinfectionsoftheskin and intestine can spread systemically, especially when left untreated, and cause pulmonary tularemia. Tularemia is an inflammatory, sepsis-like disease. Consistent with a role for NKT cells in pro-inflammatory diseases and sepsis, we discovered that Ft subspecies holarctica-derived live vaccine strain (LVS) quickly activatedlunginterstitialNKTcellsandinducedrunawayinflammation.Thus,LVS-infectedimmunecompetent micebecameseverelymorbidandsuccumbedtoatularemia-likedisease.Accordingly,NKTcell-deficientmice recovered from disease and lived despite similar bacterial burden in the lungs of both mutant and wild type mice. Survival was likely because NKT cell-deficient mice had developed the protective induced bronchus- associated lymphoid tissue (iBALT) at the peak of infection. Further, the iBALT-suppressing regulatory T cells areenrichedinwildtypemice,whilsttheiBALT-inducingIL-17-producingcells,potentiallymucosa-associated invariantTcells(MAIT),areenrichedinNKTcell-deficientmice,andviceversa.Thesenewdatasuggestthat a Treg to IL-17-producing MAIT cell imbalance underlie tularemia-like disease caused by LVS infection. Guided by these findings, we hypothesize that an Ft-derived glycolipid agonist(s) activates NKT cells in the lungs, stirring up an inflammatory milieu that prevents iBALT formation and, thereby, causing fatal tularemia- like disease in mice. To test this central hypothesis, we will elucidate the cellular and molecular mechanisms that underlie the presentation of the cytoplasmic glycolipid agonists to NKT cells and cause tularemia-like disease during a natural Ft infection (Aims 1 and 2). Further, we will elucidate and validate the chemical structure of the LVS and the type A Ft-derived glycolipid agonist(s) (Aim 3). These proposed studies will rigorouslytesttheprevailingassumptionthatDCsarecriticalforpresentingCD1d-restrictedglycolipidagonists and activating NKT cells during a natural bacterial infection. We expect to gain novel mechanisms of host interactionswithanacute,highlyvirulentpathogenicbacterium,whichcankilltheinfectedhostwithasfewas 10infectiouscells.Ourstudieswillunveilnovelinsightsintotheinteractionsbetweeninnate-likelymphocytes, e.g.,NKTcellsandMAITcells.ThesenovelinsightscoupledwiththeknowledgeoftheNKTcellagonist(s)and its/their biosynthetic pathway(s) can lead to new targets for immunotherapies and vaccine design against tularemiaandpotentiallyotheracutepulmonaryinfectiousdiseases.